Endothelial cells, the major cellular constituent of the vascular bed, provides a semi-permeable barrier between the blood and the vasculature. Loss of semi-selective endothelial cell barrier increases vascular permeability causing pulmonary edema, a cardinal feature of inflammatory vascular injury. Thus, by virtue of their unique location, the vascular endothelium, is a target for a variety of inflammatory agents and circulating leukocytes. Reactive oxygen species (ROS), generated by activated leukocytes, have been implicated in the pathophysiology of several vascular disorders including atherosclerosis and adult respiratory distress syndrome. Although the mechanisms of ROS-induced vascular disorder are not completely understood, there is increasing evidence that ROS modulate endothelial cell (EC) function by modulating signal transduction pathways and generation of second-messengers. However, ROS-mediated regulation of signal transduction in ECs is not well defined. The investigators have identified phospholipase D (PLD) activation as an important ROS-mediated signal transduction pathway in vascular ECs. Phosphatidic acid generated by the PLD activation may have profound effect on the cytoskeletal network and cellular function of the ECs. They want to test the hypothesis that ROS-mediated PLD activation involves a sequence whereby ROS stimulates tyrosine kinases and mitogen activated protein kinases (MAP kinases) which phosphorylate PLD. The investigators further hypothesize that ROS-mediated PLD activation involves small molecular weight G-proteins and actin reorganization. S.A. 1: will characterize overexpression of human PLD1 in immortalized ECs. S.A. 2: will characterize ROS-mediated PLD phosphorylation and proteins associated with PLD. S.A. 3: will characterize the role of mitogen activated protein kinases in ROS-mediated PLD activation. Finally, S.A. 4: will determine whether ROS-mediated PLD activation involves small molecular weight G-proteins. This in depth investigation of ROS-induced regulation of PLD signal transduction and actin stress fiber formation will enhance our understanding of the role of oxidative stress in vascular function under normal and pathologic conditions.